Involvement of the Anion Exchanger SLC26A6 in Prostaglandin E2- but not Forskolin-Stimulated Duodenal HCO3− Secretion
2006; Elsevier BV; Volume: 130; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2005.10.017
ISSN1528-0012
AutoresBiguang Tuo, Brigitte Riederer, Zhaohui Wang, William H Colledge, Manoocher Soleimani, Ursula Seidler,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoBackground & Aims: SLC26A6 is a recently identified apical Cl−/HCO3− exchanger with strong expression in murine duodenum. The present study was designed to examine the role of SLC26A6 in prostaglandin E2 (PGE2)-, forskolin-, and carbachol-induced duodenal HCO3− secretion. Methods: Murine duodenal mucosal HCO3− secretion was examined in vitro in Ussing chambers and mucosal SLC26A6 expression levels were analyzed by semiquantitative reverse-transcription polymerase chain reaction. Results: Basal HCO3− secretion was diminished by 20%, PGE2-stimulated HCO3− secretory response by 59%, and carbachol-stimulated response was reduced by 35% in SLC26A6−/− compared with +/+ duodenal mucosa, whereas the forskolin-stimulated HCO3− secretory response was not different. In Cl−-free solutions, PGE2- and carbachol-stimulated HCO3− secretion was reduced by 81% and 44%, respectively, whereas forskolin-stimulated HCO3− secretion was not altered significantly. PGE2 and carbachol, but not forskolin, were able to elicit a Cl−-dependent HCO3− secretory response in the absence of short-circuit current changes in cystic fibrosis transmembrane conductance regulator knockout mice. Conclusions: In murine duodenum, PGE2-mediated HCO3− secretion is strongly SLC26A6 dependent and cystic fibrosis transmembrane conductance regulator independent, whereas forskolin-stimulated HCO3− secretion is completely SLC26A6 independent and cystic fibrosis transmembrane conductance regulator dependent. Carbachol-induced secretion is less pronounced, but occurs via both transport pathways. This suggests that PGE2 and forskolin activate distinct HCO3− transport pathways in the murine duodenum. Background & Aims: SLC26A6 is a recently identified apical Cl−/HCO3− exchanger with strong expression in murine duodenum. The present study was designed to examine the role of SLC26A6 in prostaglandin E2 (PGE2)-, forskolin-, and carbachol-induced duodenal HCO3− secretion. Methods: Murine duodenal mucosal HCO3− secretion was examined in vitro in Ussing chambers and mucosal SLC26A6 expression levels were analyzed by semiquantitative reverse-transcription polymerase chain reaction. Results: Basal HCO3− secretion was diminished by 20%, PGE2-stimulated HCO3− secretory response by 59%, and carbachol-stimulated response was reduced by 35% in SLC26A6−/− compared with +/+ duodenal mucosa, whereas the forskolin-stimulated HCO3− secretory response was not different. In Cl−-free solutions, PGE2- and carbachol-stimulated HCO3− secretion was reduced by 81% and 44%, respectively, whereas forskolin-stimulated HCO3− secretion was not altered significantly. PGE2 and carbachol, but not forskolin, were able to elicit a Cl−-dependent HCO3− secretory response in the absence of short-circuit current changes in cystic fibrosis transmembrane conductance regulator knockout mice. Conclusions: In murine duodenum, PGE2-mediated HCO3− secretion is strongly SLC26A6 dependent and cystic fibrosis transmembrane conductance regulator independent, whereas forskolin-stimulated HCO3− secretion is completely SLC26A6 independent and cystic fibrosis transmembrane conductance regulator dependent. Carbachol-induced secretion is less pronounced, but occurs via both transport pathways. This suggests that PGE2 and forskolin activate distinct HCO3− transport pathways in the murine duodenum. Duodenal ulcer patients have significantly diminished proximal duodenal mucosal HCO3− secretory response to acid even after ulcer healing,1Isenberg J.I. Selling J.A. Hogan D.L. Koss M.A. Impaired proximal duodenal mucosal bicarbonate secretion in patients with duodenal ulcer.N Engl J Med. 1987; 316: 374-379Crossref PubMed Scopus (185) Google Scholar suggesting a causal role for impaired HCO3− secretion in the pathogenesis of duodenal ulcer. Experiments in animals showed that the apical membrane of duodenal enterocytes contains Cl−/HCO3− exchangers and HCO3− conductive pathways (ie, cystic fibrosis transmembrane conductance regulator [CFTR]) that are involved in duodenal HCO3− secretion.2Konturek P.C. Konturek S.J. Hahn E.G. Duodenal alkaline secretion its mechanisms and role in mucosal protection against gastric acid.Dig Liver Dis. 2004; 36: 505-512Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 3Flemstrom G. Isenberg J.I. Gastroduodenal mucosal alkaline secretion and mucosal protection.News Physiol Sci. 2001; 16: 23-28PubMed Google Scholar, 4Safsten B. Duodenal bicarbonate secretion and mucosal protection. Neurohumoral influence and transport mechanisms.Acta Physiol Scand Suppl. 1993; 613: 1-43PubMed Google Scholar, 5Brown C.D. Dunk C.R. Turnberg L.A. Cl−/HCO3− exchange and anion conductance in rat duodenal apical membrane vesicles.Am J Physiol. 1989; 257: G661-G667PubMed Google Scholar SLC26A6, also called putative anion transporter 1, is a member of a large family of anion exchangers of SLC26 (solute-linked carrier 26 gene family), which is expressed widely in kidney, pancreas, intestine, heart, muscle, and placenta.6Sterling D. Casey J.R. Bicarbonate transport proteins.Biochem Cell Biol. 2002; 80: 483-497Crossref PubMed Scopus (82) Google Scholar, 7Wang Z. Petrovic S. Mann E. Soleimani M. Identification of an apical Cl−/HCO3− exchanger in the small intestine.Am J Physiol. 2002; 282: G573-G579PubMed Google Scholar, 8Petrovic S. Ma L. Wang Z. Soleimani M. Identification of an apical Cl−/HCO3− exchanger in rat kidney proximal tubule.Am J Physiol. 2003; 285: C608-C617Crossref Scopus (52) Google Scholar, 9Alvarez B.V. Kieller D.M. Quon A.L. Markovich D. Casey J.R. Slc26a6 a cardiac chloride/hydroxyl exchanger and predominant chloride/bicarbonate exchanger of the heart.J Physiol. 2004; 561: 721-734Crossref PubMed Scopus (71) Google Scholar, 10Mount D.B. Romero M.F. The SLC26 gene family of multifunctional anion exchangers.Pflugers Arch. 2004; 447: 710-721Crossref PubMed Scopus (452) Google Scholar SLC26A6 expression is highly abundant in the duodenum and located apically on the villi of small intestine.7Wang Z. Petrovic S. Mann E. Soleimani M. Identification of an apical Cl−/HCO3− exchanger in the small intestine.Am J Physiol. 2002; 282: G573-G579PubMed Google Scholar Consequently, SLC26A6-deficient mice show a reduced basal duodenal bicarbonate secretory rate compared with wild-type littermates.11Wang Z. Wang T. Petrovic S. Tuo B. Riederer B. Barone S. Lorenz J. Seidler U. Aronson P.S. Soleimani M. Kidney and intestine transport defects in Slc26a6 null mice.Am J Physiol. 2005; 288: C957-D965Crossref PubMed Scopus (167) Google Scholar Prostaglandin E2 (PGE2) is present throughout the gastrointestinal tract and is implicated in the regulation of a variety of gastrointestinal functions including blood flow, and acid, mucus, and HCO3− secretion.12Johansson C. Bergstrom S. Prostaglandin and protection of the gastroduodenal mucosa.Scand J Gastroenterol Suppl. 1982; 77: 21-46PubMed Google Scholar Although the physiologic regulation of duodenal HCO3− secretion involves many neurohumoral factors, endogenous PGE2 is believed to be particularly important in the local control of this secretion.13Sugamoto S. Kawauch S. Furukawa O. Mimaki T.H. Takeuchi K. Role of endogenous nitric oxide and prostaglandin in duodenal bicarbonate response induced by mucosal acidification in rats.Dig Dis Sci. 2001; 46: 1208-1216Crossref PubMed Scopus (54) Google Scholar, 14Takeuchi K. Ukawa H. Kato S. 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Ichikawa A. Ushikubi F. Narumiya S. Impaired duodenal bicarbonate secretion and mucosal integrity in mice lacking prostaglandin E-receptor subtype EP3.Gastroenterology. 1999; 117: 1128-1135Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar PGE2 and its analogues, whether applied luminally or vascularly, stimulate gastroduodenal HCO3− secretion in vivo and in vitro in various species, including human beings.12Johansson C. Bergstrom S. Prostaglandin and protection of the gastroduodenal mucosa.Scand J Gastroenterol Suppl. 1982; 77: 21-46PubMed Google Scholar, 17Takeuchi K. Ukawa H. Furukawa O. Kawauchi S. Araki H. Sugimoto Y. Ishikawa A. Ushikubi F. Narumiya S. Prostaglandin E receptor subtypes involved in stimulation of gastroduodenal bicarbonate secretion in rats and mice.J Physiol Pharmacol. 1999; 50: 155-167PubMed Google Scholar, 18Takeuchi K. Yagi K. Kato S. Ukawa H. Roles of prostaglandin E-receptor subtypes in gastric and duodenal bicarbonate secretion in rats.Gastroenterology. 1997; 113: 1553-1559Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 19Yao B. Hogan D.L. Bukhave K. Koss M.A. Isenberg J.I. Bicarbonate transport by rabbit duodenum in vitro effect of vasoactive intestinal polypeptide, prostaglandin E2, and cyclic adenosine monophosphate.Gastroenterology. 1993; 104: 732-740PubMed Google Scholar, 20Mu J.Z. Hogan D.L. Koss M.A. Isenberg J.I. Theophylline and prostaglandin E2 on duodenal bicarbonate secretion role for 5′-cyclic adenosine monophosphate.Gastroenterology. 1992; 103: 153-159PubMed Google Scholar, 21Isenberg J.I. Hogan D.L. Koss M.A. Selling J.A. Human duodenal mucosal bicarbonate secretion. Evidence for basal secretion and stimulation by hydrochloric acid and a synthetic prostaglandin E1 analogue.Gastroenterology. 1986; 91: 370-378PubMed Google Scholar PGE2 stimulates adenylate cyclase activity and increases intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) levels in duodenal enterocytes,19Yao B. Hogan D.L. Bukhave K. Koss M.A. Isenberg J.I. Bicarbonate transport by rabbit duodenum in vitro effect of vasoactive intestinal polypeptide, prostaglandin E2, and cyclic adenosine monophosphate.Gastroenterology. 1993; 104: 732-740PubMed Google Scholar, 22Amelsberg M. Amelsberg A. Ainsworth M.A. Hogan D.L. Isenberg J.I. Cyclic adenosine-3′,5′-monophosphate production is greater in rabbit duodenal crypt than in villus cells.Scand J Gastroenterol. 1996; 31: 233-239Crossref PubMed Scopus (15) Google Scholar, 23Reimer R. Odes H.S. Muallem R. Schwenk M. Beil W. Sewing K.F. Cyclic adenosine monophosphate is the second messenger of prostaglandin E2- and vasoactive intestinal polypeptide-stimulated active bicarbonate secretion by guinea-pig duodenum.Scand J Gastroenterol. 1994; 29: 153-159Crossref PubMed Scopus (14) Google Scholar, 24Reimer R. Heim H.K. Muallem R. Odes H.S. Sewing K.F. Effects of EP-receptor subtype specific agonists and other prostanoids on adenylate cyclase activity of duodenal epithelial cells.Prostaglandins. 1992; 44: 485-493Crossref PubMed Scopus (39) Google Scholar but some experimental data suggest that PGE2 may stimulate duodenal HCO3− secretion by a different signal transduction pathway from that induced by cAMP.25Nyberg L. Pratha V. Hogan D.L. Rapier R.C. Koss M.A. Isenberg J.I. Human proximal duodenal alkaline secretion is mediated by Cl−/HCO3− exchange and HCO3− conductance.Dig Dis Sci. 1998; 43: 1205-1210Crossref PubMed Scopus (13) Google Scholar In rabbit proximal duodenum in vitro,19Yao B. Hogan D.L. Bukhave K. Koss M.A. Isenberg J.I. Bicarbonate transport by rabbit duodenum in vitro effect of vasoactive intestinal polypeptide, prostaglandin E2, and cyclic adenosine monophosphate.Gastroenterology. 1993; 104: 732-740PubMed Google Scholar PGE2-stimulated duodenal HCO3− secretion was abolished by luminal 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, suggesting the involvement of apical Cl−/HCO3− exchangers in the action of PGE2. Accordingly, the present study was designed to examine the role of the Cl−/HCO3− exchanger SLC26A6 and CFTR in PGE2-induced duodenal mucosal HCO3− secretion by using knockout mice lacking the SLC26A6 anion exchanger or the CFTR Cl− channel. All reagents were purchased from Sigma-Aldrich (Deisen-hofen, Germany) and Merck (Darmstadt, Germany). The mucosal solution used in the Ussing chamber experiments contained the following (in mmol/L): 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl−, 25 gluconate, and 10 mannitol. The serosal solution contained (in mmol/L): 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl−, 25 HCO3−, 2.4 HPO42−, 2.4 H2PO4−, 10 glucose, .0001 tetrodotoxin, and .0001 indomethacin. In Cl−-free solutions, Cl− was iso-osmotically replaced by gluconate. The osmolalities for both solutions were approximately 305 mOsm/kg. All studies were approved by the Hannover Medical School Committee on Investigations Involving Animals. Experiments were performed on white NMRI mice (for the Cl− substitution experiments), SLC26A6 (−/− and +/+ littermates) on a C57/B6 background, and CFTR (−/− and +/+ littermates) back-crossed for 15 generations on a NMRI genetic background. All mice were 3–5 months of age. The genotype of SLC26A6 (SLC26A6−/−) and CFTR (CFTR−/−) knockout mice were verified by polymerase chain reaction (PCR) as described previously.11Wang Z. Wang T. Petrovic S. Tuo B. Riederer B. Barone S. Lorenz J. Seidler U. Aronson P.S. Soleimani M. Kidney and intestine transport defects in Slc26a6 null mice.Am J Physiol. 2005; 288: C957-D965Crossref PubMed Scopus (167) Google Scholar, 26Seidler U. Blumenstein I. Kretz A. Viellard-Baron D. Rossmann H. Colledge W.H. Evans M. Ratcliff R. Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent HCO3− secretion.J Physiol. 1997; 505: 411-423Crossref PubMed Scopus (226) Google Scholar The mice were housed in a standard animal care room with a 12-hour light-dark cycle and were allowed free access to food and water. CFTR−/− mice and the control littermates were given electrolyte solution containing polyethylene glycol 4000 (institutional pharmacy) and fiber-free chow (diet C1013, Altromin GmbH, Lage, Lower Saxony) to prevent intestinal impaction. After brief narcosis with 100% CO2, the mice were killed by cervical dislocation. The abdomen was opened by midline incision and the proximal duodenum (a portion stretching from approximately 2 mm distal to the pylorus to the common bile duct ampulla) was removed and placed immediately in ice-cold iso-osmolar mannitol and indomethacin (1 μmol/L) solution (to suppress trauma-induced prostaglandin release). The duodenum was opened along the mesenteric border and stripped of external serosal and muscle layers by sharp dissection in the earlier-described ice-cold mannitol and indomethacin solution. The duodenal mucosa was mounted between 2 chambers with an exposed area of .625 cm2 and placed in an Ussing chamber. A parafilm O ring was used to minimize edge damage to the tissue where it was secured between the chamber halves. The mucosal side was bathed with unbuffered HCO3−-free modified Ringer's solution circulated by a gas lift with 100% O2. The serosal side was bathed with modified buffered Ringer's solution (pH 7.4) containing 25 mmol/L HCO3− and gassed with 95% O2/5% CO2. Each bath contained 10.0 mL of the respective solution maintained at 37°C by a heated water jacket. Experiments were performed under continuous short-circuited conditions to maintain the electrical potential difference at zero, except for a brief period (<2 s) at each time point when the open-circuit potential difference was measured. Luminal pH was maintained at 7.40 by the continuous infusion of 1 mmol/L HCl under the automatic control of a pH-stat system (PHM290, pH-Stat Controller; Radiometer, Copenhagen, Denmark). The volume of the titrant infused per unit time was used to quantitate HCO3− secretion. These measurements were recorded at 5-minute intervals. The rate of luminal HCO3− secretion is expressed as μmol/cm2/h1. Transepithelial short-circuit current (Isc) (reported as μeq/cm2/h1) and potential difference (expressed as mV) were measured via an automatic voltage clamp (Voltage-Current Clamp, EVC-4000; World Precision Instruments, Berlin, Germany). Transepithelial resistance (reported as ω · cm2) was calculated according to Isc values and potential difference values. After a 30-minute measurement of basal parameters, stimulants (PGE210−6 mol/L, forskolin 10−5 mol/L, carbachol 10−4 mol/L) were added to the serosal side of tissue in Ussing chambers. Changes in duodenal bicarbonate secretion, Isc, and potential difference during the 40-minute period ensuing after the addition of stimulants were determined. The expression of SLC26A6 messenger RNA (mRNA) in duodenum of CFTR knockout mice and their wild-type littermates was determined by semiquantitative reverse-transcription PCR. Segments of duodenal mucosae were dissected free of seromuscular layers as described earlier for Ussing chamber experiments. Total RNA was extracted as described previously by Chomczynski and Sacchi.27Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (66799) Google Scholar Homologous primers for murine SLC26A6 were designed from the published sequence SLC26A6 (NM_134420). The SLC26A6 forward primer was 5′-CCAGCAATCAAGAAGATGCC-3′ and the reverse primer was 5′-ACACTTCCACTTCAATCTCCC-3′. Semiquantitative reverse-transcription PCR was performed as described previously in Rossmann et al,28Rossmann H. Bachmann O. Vieillard-Baron D. Gregor M. Seidler U. Na+/HCO3− cotransport and expression of NBC1 and NBC2 in rabbit gastric parietal and mucous cells.Gastroenterology. 1999; 116: 1389-1398Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar and cytokeratin 18 was used as an internal standard29Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. The catalog of human cytokeratins patterns of expression in normal epithelia, tumors and cultured cells.Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4691) Google Scholar because both proteins are expressed in the surface epithelium. The ethidium bromide–stained bands were digitized by the Kodak Digital Science Image Station 440CF (Kodak, Stuttgart, Baden-Württemberg) and their optical density was measured by TotalLab software (Nonlinear Dynamics, Newcastle Upon Tyne, England). Primers for cytokeratin 18 were deduced from the published sequence Krt1-18 (NM_010664) (forward primer: 5′-GGACTCCATGAAAAACCAGAAC-3′, reverse primer: 5′-TCTGCCATCCACGATCTTAC-3′). These primers correspond to nt 963–984 and to nt 1284–1265 of the published mRNA sequence. The PCR product was approximately 322 bp and was verified by sequencing. All results are expressed as means ± SEM. ΔJHCO3− and ΔIsc refer to stimulated peak responses minus basal levels. Basal values for bicarbonate and Isc refer to an average taken over a 30-minute baseline period. Data were analyzed by Student t tests or by 1-way analysis of variance (ANOVA) with the Student–Newman–Keul post hoc test. A P value of less than .05 was considered statistically significant. In SLC26A6 knockout mice, basal duodenal HCO3− secretion was reduced significantly by 20% (SLC26A6−/−: .920 ± .065 μMol/cm2h, n = 9 vs SLC26A6+/+: 1.139 ± .076 μMol/cm2h, n = 9; P < .05), whereas basal Isc was not altered significantly (SLC26A6−/−: 1.497 ± .206 μEq/cm2h, n = 9 vs SLC26A6+/+: 1.472 ± .212 μEq/cm2h, n = 9; P > .05). The net maximal increase in the HCO3− secretory rate (ΔJHCO3−) induced by PGE2 was reduced by 59% compared with wild-type littermates (Figure 1A). No significant change in PGE2-stimulated ΔIsc in SLC26A6−/− mice was noted (Figure 1B). In contrast, forskolin-stimulated duodenal ΔJHCO3− and ΔIsc were not altered significantly in SLC26A6−/− mice (Figure 1). Carbachol, on the other hand, stimulated HCO3− secretion to a lesser degree in SLC26A6−/− compared with +/+ mice, but the reduction (35%) in ΔJHCO3- was not nearly as large as that seen for PGE2 stimulation (Figure 1A). Carbachol-stimulated ΔIsc was unaltered in the absence of SLC26A6 (Figure 1B). The results suggest that the loss of the apical anion exchanger SLC26A6 interferes with maximal stimulation of HCO3− secretion by PGE2 and carbachol, but not by forskolin. To confirm the involvement of the Cl−/HCO3− exchanger in PGE2-mediated HCO3− secretion, we studied its effect in the absence of Cl− either bilaterally or luminally in white Swiss mice. In bilateral Cl−-free solutions, both basal duodenal HCO3− secretion (Cl−-free: .919 ± .079 μMol/cm2-h, n = 10 vs Cl−-containing: 1.223 ± .082 μMol/cm2-h, n = 11; P < .05) and Isc (Cl−-free: 1.723 ± .189 μEq/cm2-h, n = 10 vs Cl−-containing: 2.276 ± .271 μEq/cm2-h, n = 11; P < .05) were lower than those in Cl−-containing, and PGE2-stimulated duodenal ΔJHCO3− was decreased considerably by 81% (Figure 2A). Of course, PGE2-induced ΔIsc (which is carried largely by Cl− ions) was very low (Figure 2B). Although forskolin-stimulated ΔIsc also was reduced by 69% in bilateral Cl−-free solutions (Figure 2B), forskolin-stimulated duodenal ΔJHCO3− was not decreased significantly (Figure 2A). Carbachol-mediated ΔJHCO3− was reduced by 44% (Figure 2A) in bilateral Cl−-free solutions and therefore showed less dependence on the presence of Cl− than PGE2-mediated HCO3− secretion, but more than forskolin-stimulated secretion. Likewise, carbachol-induced ΔIsc also was very low in bilateral Cl−-free solutions (Figure 2B). When Cl− was removed from the luminal bath only, PGE2-stimulated ΔJHCO3− also was reduced strongly by 69% compared with controls (Figure 3A), but PGE2-stimulated Isc response was not altered significantly (Figure 3B). Forskolin-stimulated HCO3− and Isc were not changed (data not shown). This strongly suggests that a Cl−/HCO3− exchange process is the major pathway for PGE2-stimulated, but not for forskolin-stimulated, HCO3− secretion. cAMP-dependent stimulation of HCO3− secretion is believed to be mediated via the CFTR anion channel,26Seidler U. Blumenstein I. Kretz A. Viellard-Baron D. Rossmann H. Colledge W.H. Evans M. Ratcliff R. Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent HCO3− secretion.J Physiol. 1997; 505: 411-423Crossref PubMed Scopus (226) Google Scholar, 30Clarke L.L. Harline M.C. Dual role of CFTR in cAMP-stimulated HCO3− secretion across murine duodenum.Am J Physiol. 1998; 274: G718-G726PubMed Google Scholar, 31Hogan D.L. Crombie D.L. Isenberg J.I. Svendsen P. Schaffalitzky de Muckadell O.B. Ainsworth M.A. CFTR mediates cAMP- and Ca2+-activated duodenal epithelial HCO3− secretion.Am J Physiol. 1997; 272: G872-G878PubMed Google Scholar and both PGE2 and forskolin stimulate an increase in duodenal enterocyte cAMP levels.22Amelsberg M. Amelsberg A. Ainsworth M.A. Hogan D.L. Isenberg J.I. Cyclic adenosine-3′,5′-monophosphate production is greater in rabbit duodenal crypt than in villus cells.Scand J Gastroenterol. 1996; 31: 233-239Crossref PubMed Scopus (15) Google Scholar The earlier-described results, however, suggest a profound difference in the transport pathways for PGE2 vs forskolin-stimulated HCO3− secretion. To gain further insight into the reason for this difference, we studied PGE2 and forskolin-stimulated HCO3− secretion and Isc in the duodenum of CFTR-deficient mice. As previously observed,26Seidler U. Blumenstein I. Kretz A. Viellard-Baron D. Rossmann H. Colledge W.H. Evans M. Ratcliff R. Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent HCO3− secretion.J Physiol. 1997; 505: 411-423Crossref PubMed Scopus (226) Google Scholar basal duodenal HCO3− secretion and Isc in CFTR knockout mice (CFTR−/−) were reduced by 37% and 44%, respectively, compared with wild-type littermates (CFTR+/+) (Figure 4). Bilateral Cl− removal reduced basal HCO3− secretion in CFTR knockout mice even further (by 51%) (Figure 4A), indicating that both the CFTR channel and the Cl−/HCO3− exchanger contribute to basal HCO3− secretion. Surprisingly, PGE2 markedly stimulated duodenal HCO3− secretion without any effect on Isc in CFTR−/− mice, and the ΔJHCO3− induced by PGE2 reached 71% of the secretion observed in CFTR+/+, but it was lower than in CFTR+/+ (Figure 5). However, after bilateral Cl− removal, PGE2 did not significantly stimulate duodenal HCO3− secretion in CFTR−/− mice (Figure 5A), suggesting that PGE2-stimulated HCO3− secretion in CFTR knockout mice is mediated by Cl−/HCO3− exchange. Likewise, carbachol stimulated duodenal HCO3− secretion without any effect on Isc in CFTR knockout mice by 48% of the HCO3− secretory response in CFTR+/+ (Figure 5), suggesting that carbachol also is able to stimulate electroneutral HCO3− secretion in the absence of CFTR activation. In contrast, forskolin did not stimulate duodenal HCO3− secretion and Isc in CFTR−/− tissue (Figure 5).Figure 5Effects of PGE2 (10−6 mol/L), forskolin (10−5 mol/L), and carbachol (10−4 mol/L) on (A) duodenal HCO3− secretion and (B) Isc in CFTR knockout mice (CFTR−/−) and their wild-type littermates (CFTR+/+). The data shown represent the net maximal increases in HCO3− secretion and Isc observed, and were expressed as the mean ± SEM. In CFTR−/− duodenum, PGE2 stimulated ΔJHCO3− by 71% of the response observed in CFTR+/+ tissue and carbachol stimulated ΔJHCO3− by 48% of the response observed in CFTR+/+ tissue, but both had no significant effects on Isc. After bilateral Cl− removal, PGE2 did not stimulate HCO3− secretion significantly in CFTR−/−. In contrast, forskolin did not significantly stimulate HCO3− and Isc in CFTR−/− (compared with CFTR+/+: *P < .05; ***P < .001; ****P < .0001; compared with CFTR−/− + Cl−-free: a***P < .001, by 1-way ANOVA with the Student–Newman–Keul post hoc test in the PGE2 group, by Student t tests in other groups). ■, CFTR+/+ , CFTR−/−; , CFTR−/− and Cl− free.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Changes in HCO3− secretion potentially could be secondary to an increased conductance of the paracellular shunt pathway. Table 1 shows the transepithelial resistance values before the addition of PGE2 and the maximal value that we measured after adding PGE2 (peak at 15 to 20 minutes after administration of PGE2, which was consistent with the peak response of HCO3− secretion). The data show that the addition of PGE2 resulted in a decrease in tissue conductance under any observed condition. Although the decrease in conductance was higher in tissues with higher secretory rates, it was also to some extent seen in tissues that did not secrete Cl− and HCO3− (eg, CFTR−/− and Cl−-free). Our results are consistent with previous observations in rabbit duodenum32Macherey H.J. Petersen K.U. Rapid decrease in electrical conductance of mammalian duodenal mucosa in vitro Combined effects of prostaglandin E2 and bicarbonate.Gastroenterology. 1989; 97: 1448-1460PubMed Google Scholar and indicate that PGE2–mediated HCO3− secretion is not likely mediated by an increase in paracellular permeability of duodenal mucosa.Table 1Effects of PGE2 on Transepithelial Resistance of Duodenal MucosaBasal TER (ω.cm2)Peak TER (ω.cm2)SLC26A6+/+ (N = 9)24.79 ± 2.4230.59 ± 2.81aP < .0001;SLC26A6 −/− (N = 9)22.58 ± 2.0227.20 ± 2.61aP < .0001;Cl− containing (N = 11)26.58 ± 2.2432.99 ± 2.36aP < .0001;Both Cl− free (N = 10)30.30 ± 2.5035.87 ± 3.49bP < .01;Luminal Cl− free (N = 11)27.71 ± 2.1233.27 ± 2.83cP < .001;CFTR+/+ (N = 8)23.48 ± 2.2828.62 ± 2.60cP < .001;CFTR −/− (N = 7)24.07 ± 2.2026.41 ± 2.67dP < .05; compared with basal values.CFTR−/− + both Cl− free (N = 6)29.27 ± 2.5633.41 ± 2.48dP < .05; compared with basal values.NOTE. Values are from the same experiments on effects of PGE2 on bicarbonate secretion and Isc, and are expressed as the mean ± SEM. Basal TER refers to the values before addition of PGE2 Peak TER refers to the maximal increase after addition of PGE2 (which was consistent with the peak response of HCO3− secretion). Data were analyzed by paired t test. TER, transepithelial resistance.a P < .0001;b P < .01;c P < .001;d P < .05; compared with basal values. Open table in a new tab NOTE. Values are from the same experiments on effects of PGE2 on bicarbonate secretion and Isc, and are expressed as the mean ± SEM. Basal TER refers to the values before addition of PGE2 Peak TER refers to the maximal increase after addition of PGE2 (which was consistent with the peak response of HCO3− secretion). Data were analyzed by paired t test. TER, transepithelial resistance. The earlier-described data indicate that PGE2-induced HCO3− secretion largely is SLC26A6 dependent and CFTR independent. One important piece of information is whether SLC26A6 expression is altered in the absence of CFTR expression. We therefore measured the expressions of SLC26A6 in duodenal mucosae of CFTR−/− mice and +/+ littermates. Figure 6 shows that the expression level of SLC26A6 was not altered in CFTR knockout mice compared with wild-type littermates (P > .05), indicating that the CFTR gene defect does not induce down-regulation of SLC26A6. This study confirms that the recently cloned anion exchange protein SLC26A6 co
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